This invention relates to a process for measuring the activity of a dehydrogenase and a process for determining the amount of a substrate using a dehydrogenase acting specifically thereupon.
The activity of dehydrogenases such as lactate dehydrogenase (hereinafter referred to as "LDH"), glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, .alpha.-hydroxybutyrate dehydrogenase (hereinafter referred to as ".alpha.-HBD"), a 3.alpha.-hydroxysteroid dehydrogenase (hereinafter referred to as "3.alpha.-HSD"), etc., using nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotidephosphate (NADP) as hydrogen acceptor can be measured by a UV method or a visible colorimetric method. According to the UV method, the increase in absorbance due to NADH (nicotinamide adenine dinucleotide, reduced form) or NADPH (nicotinamide adenine dinucleotidephosphate, reduced form) produced by the enzymatic reaction of a substrate and a dehydrogenase is usually measured at 340 nm in a certain time period (employing a so-called rate assay).
According to the visible colorimetric method, the produced NADH or NADPH reduces a tetrazolium salt with the aid of an electron carrier or diaphorase to produce a formazan dye, the color of which is measured colorimetrically. The principle of the visible colorimetric method, for example, in the measurement of the activity of LDH can be shown as follows: ##STR1##
Since these reactions proceed quantitatively and specifically, the activity of LDH can be measured by measuring quantitatively the color concentration of the formazan produced. Generally speaking, the visible colorimetric method comprises conducting an enzymatic reaction in a certain number of minutes, adding an enzymatic reaction stopper to the reaction system to stop the reaction, and measuring the amount of dye corresponding to the amount of NADH produced colorimetrically.
As a method for stopping the reaction, there is most popularly employed a method wherein the enzymatic reaction is stopped by making the reaction solution strongly acidic. As the acidifying agent, there is generally used an inorganic acid such as hydrochloric acid. But the use of such an inorganic acid produces various disadvantages in that the formazan (or diformazan) dye produced is easily faded when exposed to light, some formazans produce clouding or precipitation due to low solubility, staining of test tubes and cuvettes takes place thus causing errors in measurement, a large specimen blank value due to clouding is obtained in the case of using chyle serum as specimen, which cause errors in ordinary examinations wherein no specimen blank is taken, and the like. Therefore, elimination of such disadvantages has been desired.